Screening for neurotoxic amino acid associated with neurological disorders

ABSTRACT

Methods for screening for neurological disorders are disclosed. Specifically, methods are disclosed for screening for neurological disorders in a subject by analyzing a tissue sample obtained from the subject for the presence of elevated levels of neurotoxic amino acids or neurotoxic derivatives thereof associated with neurological disorders. In particular, methods are disclosed for diagnosing a neurological disorder in a subject, or predicting the likelihood of developing a neurological disorder in a subject, by determining the levels of β-N-methylamino-L-alanine (BMAA) in a tissue sample obtained from the subject. Methods for screening for environmental factors associated with neurological disorders are disclosed. Methods for inhibiting, treating or preventing neurological disorders are disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/731,411, filed Dec. 8, 2003, now U.S. Pat. No. 7,256,002 B2, issuedAug. 14, 2007, which claims the benefit of U.S. Provisional PatentApplication No. 60/494,686, filed Aug. 13, 2003.

FIELD OF THE INVENTION

The present invention relates to screening for neurological disorders.Specifically, the invention relates to screening for neurologicaldisorders in a subject by analyzing a tissue sample from the subject todetermine the presence of neurotoxic amino acids or neurotoxicderivatives thereof associated with neurological disorders. Inparticular, the present invention relates to methods for diagnosing aneurological disorder in a subject, or predicting the likelihood ofdeveloping a neurological disorder in a subject, by determining thelevels of β-N-methylamino-L-alanine (BMAA) or a neurotoxic derivativethereof, in a tissue sample obtained from the subject. Further, theinvention relates to screening environmental samples for a neurotoxicamino acid or neurotoxic derivative thereof associated with neurologicaldisorders. Further, the invention relates to inhibiting neurologicaldisorders.

BACKGROUND OF THE INVENTION

A unique neurological disease initially identified among the Chamorropeople of Guam by Kurland and Mulder (1954) is characterized by acombination of symptoms including stooped posture, a blankexpressionless face, dementia, slow shuffling movement, a resting tremorthat stops upon deliberate action, slow movements, and muscle atrophythat results in muscles dipping down in the hand. In some clinicalmanifestations, patients have clinical symptoms indistinguishable fromamyotrophic lateral sclerosis (ALS). Other patients have Parkinsonismfeatures combined with dementia (Parkinsonism Dementia Complex, PDC). Instill others, only dementia is observed. Some patients also have bothALS and PDC. Neuropathologically, all clinical forms of the diseaseresult in a specific feature, neurofibrillary tangles, found in thecortex and in the spinal cord. Because the disease has aspects thatresemble amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD)and Alzheimer's disease (AD), this disease is known as amyotrophiclateral sclerosis-Parkinsonism dementia complex of Guam (ALS-PDC) and isalso known as lytico-bodig.

SUMMARY OF THE INVENTION

The present invention provides methods of screening a subject having orat risk of having a neurological disorder by analyzing a tissue samplefrom the subject to determine the presence of a neurotoxic amino acid,or neurotoxic derivative thereof, associated with the neurologicaldisorder. The neurotoxic amino acid or neurotoxic derivative thereof canbe a glutamate receptor agonist such as β-N-methylamino-L-alanine(BMAA), or β-N-oxalyl-amino-L-alanine (BOAA). In a tissue sample,protein-bound neurotoxic amino acid or neurotoxic derivative thereof canbe analyzed, free (unbound) neurotoxic amino acid or neurotoxicderivative thereof can be analyzed, or both protein-bound and freeneurotoxic amino acid or neurotoxic derivative thereof can be analyzedin a sample. In a tissue sample, protein-bound BMAA, free BMAA, or bothprotein-bound BMAA and free BMAA can be analyzed. The subject may havesymptoms of a neurological disorder, or may be asymptomatic for aneurological disorder, or may have been identified as being at risk fordeveloping a neurological disorder. The neurotoxic derivative may be anyderivative having neurotoxic activity, such as a carbamate adduct ormetabolite of the neurotoxic amino acid.

The present invention provides methods of screening a subject having orat risk of having a neurological disorder by analyzing a tissue samplefrom the subject to determine the presence of a neurotoxic amino acid orneurotoxic derivative thereof associated with the neurological disorder,wherein the presence of a detectable level of a neurotoxic amino acid orneurotoxic derivative thereof indicates a neurological disorder. Methodsof the invention can be used to detect neurological disorders includinga neurofibrillary tangle disorder (NFT disorder) such as amyotrophiclateral sclerosis-Parkinsonism dementia complex (ALS-PDC), Alzheimer'sdisease, or progressive supranuclear palsy (PSP), a movement disordersuch as Parkinson's disease, or a motor neuron disease such asamyotrophic lateral sclerosis (ALS).

The present invention provides methods of screening a subject having orat risk of having a neurological disorder by analyzing a tissue samplefrom the subject to determine the presence of a neurotoxic amino acid orneurotoxic derivative thereof associated with the neurological disorder,wherein the methods can be used to predict the likelihood of developinga neurological disease, and/or to predict the latency period prior toonset of the neurological disorder, and/or to predict the severity ofthe neurological disorder. Methods of the present invention can bepracticed using tissue samples including, but not limited to,neurological tissue or non-neurological tissue. Neurological tissue canbe associated with the central nervous system (CNS), including braintissue or cerebral-spinal fluid (CSF), or may be associated with theperipheral nervous system (PNS). Non-neurological tissue can bekeratinous tissue including but not limited to, hair, skin, nail,including fingernail or toenail, feather, claw, hoof, or horn.Non-neurological tissue can be non-keratinous tissue including but notlimited to, bood, serum, saliva, or urine.

The present invention provides methods for screening an environmentalsample to determine if the environmental sample is associated with aneurological disorder, by analyzing the environmental sample todetermine the presence of a neurotoxic amino acid or neurotoxicderivative thereof associated with the neurological disorder. Theneurotoxic amino acid or neurotoxic derivative thereof can be aglutamate receptor agonist such as a methylated alanine, in particular,BMAA. Suitable environmental samples include water and/or food items orsources.

The present invention provides methods for screening an environmentalsample to determine if the sample is associated with a neurologicaldisorder, by detecting neurotoxic amino acid or neurotoxic derivativethereof producing cyanobacteria in the environmental sample. Theneurotoxic amino acid or neurotoxic derivative thereof can be aglutamate receptor agonist such as a methylated alanine, in particular,BMAA. Methods of the invention are suitable for detecting cyanobacteriaproducing the neurotoxic amino acid or neurotoxic derivative thereof,including cyanobacteria of the genus Nostoc and/or Anabena. Suitableenvironmental samples include water and/or food items or sources.

The present invention provides methods for inhibiting a neurologicaldisorder in a subject by reducing levels of a neurotoxic amino acid orneurotoxic derivative thereof associated with the neurological disorder,in particular by releasing the neurotoxic amino acid or neurotoxicderivative thereof from an endogenous reservoir. The neurotoxic aminoacid or neurotoxic derivative thereof can be a glutamate receptoragonist such as a methylated alanine, in particular, BMAA.

The present invention provides methods inhibiting a neurologicaldisorder in a subject by increasing the cellular concentration of aneuroprotectant compound that blocks interaction of a neurotoxic aminoacid or neurotoxic derivative thereof associated with the neurologicaldisorder with a target molecule. The neurotoxic amino acid or neurotoxicderivative thereof can be a glutamate receptor agonist such as amethylated alanine, in particular, BMAA. The neuroprotectant compoundcan be glutamic acid. An agent that binds or chelates the neurotoxicamino acid or neurotoxic derivative thereof, can be included.

The present invention further provides kits for screening a subjecthaving or at risk of having a neurological disorder, wherein the kitsinclude a means for obtaining a tissue sample from the subject and ameans for analyzing the tissue sample to determine the presence of aneurotoxic amino acid or neurotoxic derivative thereof associated withthe neurological disorder. The kit may include means for determining thepresence of a glutamate receptor agonist such as a methylated alanine,in particular BMAA. The kit may include means for analyzingprotein-bound BMAA, free BMAA, or both protein-bound BMAA and free BMAAin the sample. The kit may include means for obtaining and analyzing aplurality of tissue samples from the subject. The tissue samples mayinclude a sample of a tissue in which a neurotoxic amino acid orneurotoxic derivative thereof is known to accumulate and a sample of atissue in which neurotoxic amino acid or neurotoxic derivative thereofis known to not accumulate. The tissue samples may include a sample ofat least two distinct tissues in which a neurotoxic amino acid orneurotoxic derivative thereof is known to accumulate. The kit mayinclude means for performing repeated screening of the subject.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows concentrations of BMAA and glutamic acid (GLU) in Cycasmicronesia Hill, normalized by dividing the maximum concentration ofeach amino acid, which permits a comparison of relative abundancethroughout the plant; values below 9 μg/g cannot be seen in this FIGUREas these values are too small relative to the maximum concentration.

Table 1 shows BMAA and GLU concentrations in various tissues of Cycasmicronesia Hill; concentrations are expressed as μg/g.

Table 2 shows BMAA concentrations in samples of cycad tissues, cyadflour, and flying fox tissues.

Table 3 shows levels of free and protein-associated BMAA in tissuesamples from the superior frontal gyrus of patients from Chamorro andCanadian populations.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods for screening for neurologicaldisorders. Methods as provided herein can be used to diagnose or predictneurological disorders in a subject, to screen for environmental factorsassociated with neurological disorders, and to inhibit neurologicaldisorders in a subject.

The present invention provides methods for screening a subject forneurological disorders by analyzing a tissue sample from the subject todetermine the presence of a neurotoxic amino acid or neurotoxicderivative thereof associated with the neurological disorder. Thepresent invention further provides methods for screening environmentalsamples to determine the presence of a neurotoxic amino acid orneurotoxic derivative thereof associated with neurological disorders.The phrase “to determine the presence of a neurotoxic amino acid orneurotoxic derivative thereof” or “determining the presence of aneurotoxic amino acid or neurotoxic derivative thereof” or a similarphrase, includes not only determining the presence or absence ofdetectable levels of a neurotoxic amino acid or neurotoxic derivativethereof, but also includes quantifying the levels of a neurotoxic aminoacid or neurotoxic derivative thereof detected in a sample. Thus, in aparticular embodiment, “determining the presence of a neurotoxic aminoacid or neurotoxic derivative thereof” in a sample can includedetermining the level of the neurotoxic amino acid or neurotoxicderivative thereof and can further include determining whether the levelof neurotoxic amino acid or neurotoxic derivative thereof in the sampleis elevated or decreased in comparison with the levels detected in othersamples.

Screening includes but is not limited to, diagnosing or predictingneurological disorders in a subject by analyzing a tissue sample fromthe subject. Screening may be carried out on a subject having aneurological disorder, or may be carried out on a subject at risk ofhaving a neurological disorder, or may be carried out on a subjecthaving no known risk of having a neurological disorder. Screeningfurther includes analyzing environmental samples to determine actual orpotential exposure of a subject to a neurotoxic amino acid or neurotoxicderivative thereof associated with a neurological disorder.

As provided herein, neurotoxic amino acids or neurotoxic derivativesthereof associated with neurological disorders include, but are notlimited to, non-protein amino acids, excitatory amino acids, amino acidanalogs, amino acid metabolites, carbamate adducts of amino acids, andconjugates of amino acids. In one embodiment, one or more neurologicaldisorders can be screened for in a subject by determining the presenceof β-N-methylamino-L-alanine (BMAA) in a sample of tissue obtained fromthe subject. In another embodiment, one or more neurological disorderscan be screened for by determining the presence of(S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) ina sample of tissue obtained from the subject. In yet another embodiment,one or more neurological disorders can be screened for in a subject bydetermining the presence of β-N-oxalyl-amino-L-alanine (BOAA, alsodescribed as S-(−)-β-N-oxalyl-β-diaminopropionic acid) in a sample oftissue obtained from the subject. It is understood that methods fordetermining of neurotoxic amino acid or neurotoxic derivative thereofinclude, when necessary, methods for distinguishing the neurotoxicisomer from the nonneurotoxc isomer of the same compounds, e.g., fordistinguishing neurotoxic L-BOAA from non-neurotoxic D-BOAA.

Neurotoxic amino acids of the present invention can be non-protein aminoacids including but not limited to, β-alanine (3-alanine),4-aminobutyrate (GABA), 3-cyanoalanine (β-cyanoalanine), 2-aminobutyricacid, 2-methylene-4-aminobutyric acid, 3-methylene-4-aminobutyric acid,2-aminoisobutyric acid, 5-aminolevulinic acid, 2-amino-4-methylhexanoicacid (homoisoleucine), 2-amino-4-methylhex-4-enoic acid,2-amino-4-methylhex-5-ynoic acid, 2-amino-3-methylpentanoic acid,2-aminoadipic acid, 4-ethylideneglutamic acid, 3-aminoglutaric acid,2-aminopimelic acid, N4-ethylasparagine, N4-methylasparagine,erythro-4-methylglutamic acid, 4-methyleneglutamic acid.4-methyleneglutamine, N5-methylglutamine, N5-ethylglutamine (theanine),N5-isopropylglutamine, 2-amino-4-(aminoxy)butyric acid (canaline),2,4-diaminobutyrate, N4-acetyl-2,4-diaminobutyrate,N4-lactyl-2,4-diaminobutyrate, N4-oxalyl-2,4-diaminobutyrate,2,3-diaminopropionic acid, N3-acetyl-2,3-diaminopropionic acid,N3-methyl-2,3-diaminopropionic acid, N3-oxalyl-2,3-diaminopropionicacid, N6-acetyllysine, N6-methyllysine, N6-trimethyllysine (laminine),ornithine (2,5-diaminopentanoic acid), saccharopine(N6-(2′-glutamyl)lysine, 2,6-diaminopimelic acid,N4-(2-hydroxylethyl)asparagine, erythro-3-hydroxyaspartic acid,4-hydroxyarginine, 4-hydroxycitrulline, threo-4-hydroxyglutarnic acid,3,4-dihydroxyglutamic acid, 3-hydroxy-4-methylglutamic acid,3-hydroxy-4-methyleneglutamic acid, 4-hydroxy-4-methylglutamic acid,4-hydroxy-glutamine, N5-(2-hydroxyethyl)glutamine, 5-hydroxynorleucine,threo-4-hydroxyhomoarginine, homoserine, O-acetylhomoserine,O-oxalylhomoserine, O-phosphohomoserine, 4-hydroxyisoleucine,5-hydroxymethylhomocysteine, threo-3-hydroxyleucine, 5-hydroxyleucine,2-hydroxylysine, 4-hydroxylysine, 5-hydroxylysine,N6-acetyl-5-hydroxylysine, N6-trimethyl-5-hydroxylysine,4-hydroxyornithine, mimosine, 4-hydroxynorvaline, 5-hydroxynorvaline,2-amino-4,5-dihydroxypentanoic acid, 2-amino-4-hydroxypimelic acid,4-hydroxyvaline, O-acetylserine, O-phosphoserine, pipecolic acid(piperidine-2-carboxylic acid), 3-hydroxypipecolic acid,trans-4-hydroxypipecolic acid, trans-5-hydroxypipecolic acid,5-hydroxy-6-methylpipecolic acid, 4,5-dihydroxypipecolic acid,trans-3-hydroxyproline, trans-4-hydroxyproline,trans-4-hydroxymethylproline, azetidine-2-carboxylic acid,N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid,4,5-dehydropipecolic acid (baikiain), 3-amino-3-carboxypyrrolidone(cucurbitine), 2-(cyclopent-2′-enyl)glycine, 5-hydroxytryptophan,albizziine (2-amino-3-ureidopropionic acid), arginosuccinic acid,canavinosuccinic acid, citrulline, homoarginine, homocitrulline,indospicine, O-ureidohomoserine, 6-hydroxykynurenine,3-(4-aminophenyl)alanine, 3-(3-aminomethylphenyl)alanine,3-(3-carboxyphenyl)alanine, 3-carboxytyrosine,3-(3-hydroxymethylphenyl)alanine, 3-(3-hydroxyphenyl)alanine,3-(3,4-dihydroxyphenyl)alanine (L-DOPA), 2-(phenyl)glycine,2-(3-carboxyphenyl)glycine, 2-(3-carboxy-4-hydroxyphenyl)glycine,2-(3-hydroxyphenyl)glycine, 2-(3,5-dihydroxyphenyl)glycine,4-aminopipecolic acid, guvacine,2-amino-4-(isoxazolin-5-one)-2-yl)butyric acid, lathyrine, ortetrahydrolathyrine. (Spencer and Berman, 2003, in, Plant Toxins andHuman Health, CABI, pp 1-23). The present disclosure provides sufficientguidance for one of skill in the art to identify a neurotoxicnon-protein amino acid of the present invention.

Neurotoxic derivatives of non-protein amino acids include but are notlimited to metabolites, carabamate adducts, analogs, and other aminoacid derivatives having neurotoxic activity. In accordance with oneaspect, neurotoxic derivatives are carbamate adducts neurotoxicderivative thereof associated with neurological disorders, e.g., bydraining endogenous reservoirs of the neurotoxic amino acid orneurotoxic derivative thereof. Inhibiting includes, but is not limitedto, treating existing neurological disorders or preventing neurologicaldisorders. In one embodiment, the present invention provides methods fordraining endogenous reservoirs of BMAA or derivatives thereof in asubject.

In accordance with another aspect, the invention provides methods forinhibiting a neurological disorder in a subject by interfering with theinteraction between a neurotoxic amino acid or neurotoxic derivativethereof and its target molecule. In particular, the invention providesmethods for inhibiting a neurological disorder by increasing thecellular concentration of a neuroprotectant compound that blocksinteraction of a neurotoxic amino acid or neurotoxic derivative thereofwith a target molecule. In one embodiment, the neurotoxic amino acid orneurotoxic derivative thereof is BMAA or a BMAA derivative, and theneuroprotectant compound is glutamic acid or a glutamic acid analog.Agents that bind neurotoxic amino acids or neurotoxic derivativesthereof can be included to sequester neurotoxic amino acid or neurotoxicderivative thereof released from endogenous reservoirs. Chelating agentscan be included to chelate metal ions released when neurotoxic aminoacids or neurotoxic derivatives thereof are released from endogenousreservoirs.

As provided herein, a subject may be any organism suitable forpracticing the methods of the present invention. In particular, asubject is a mammal, more particularly a primate, even more particularlya human. In one embodiment, a subject is an experimental animal that isexposed to a neurotoxic amino acid or neurotoxic derivative thereofassociated with neurological disorders. Such experimental animalsinclude, but are not limited to, a mouse, rabbit, rat, bat, pig, sheep,cow, monkey, ape, or other animal suitable for research on neurologicaldisorders. In one embodiment, methods of the present invention arecarried out using an experimental animal for which an animal model ofone or more neurological diseases exists. In another embodiment, methodsof the present invention are carried out using an experimental animal aspart of developing an animal model of one or more neurological diseases.In yet another embodiment, methods of the present invention are carriedout using an experimental animal in which the effects of exposure to aneurotoxic amino acid or neurotoxic derivative thereof associated withneurological disorders are measured by studies of brain chemistry,structure, or function. In one (carbamates) of neurotoxic amino acids.In one embodiment, neurotoxic derivatives of the present invention arecarbamate adducts of BMAA, includingα-N-carboxy-β-N-methylamino-L-alanine (BMAA-α-NCO₂) and/orβ-(N-carboxy-N-methyl)-amino-L-alanine (BMAA-β-NCO₂), (Brownson et al.,2002, J Ethnopharmacol 82: 159-167; Myers and Nelson, 1990, J Biol Chem265:10193-10195). In accordance with another aspect, neurotoxicderivatives include the neurotoxic isomer of a neurotoxic amino acid,although it could alternately be understood that the neurotoxic isomeris the neurotoxic amino acid in a particular embodiment. Neurotoxicderivatives may also be methylated, carbamylated, or hydroxylatedmetabolites, or metabolites conjugated to sugars, lipids, or proteins.It is understood that the methods provided herein are suitable fordetermining neurotoxins associated with neurological disorders, and mayprovide a robust measurement of neurotoxin even when the compound beingmeasured is not necessarily the compound or compounds acting in vivo ina particular subject. In one embodiment, the present disclosure providesmethods for determining BMAA levels in tissue samples and environmentalsamples, and these methods generate robust results even when thesemethods do not distinguish whether BMAA, or a derivative such as acarbamate adduct of BMAA (e.g., (BMAA-α-NCO₂ orβ-(N-carboxy-N-methyl)-amino-L-alanine (BMAA-β-NCO₂) is the most activecompound in a particular embodiment. The methods presented herein arerobust, and can be further refined by one of skill in the art, accordingto the particular circumstances of a particular embodiment.

In accordance with another aspect, the present invention providesmethods for screening environmental samples for neurotoxic amino acidsor neurotoxic derivatives thereof associated with neurologicaldisorders. Screening environmental samples for neurotoxic amino acids orneurotoxic derivatives thereof includes, but is not limited to,screening to determine actual or potential exposure of a subject toneurotoxic amino acids or neurotoxic derivatives thereof associated withneurological disorders, and screening to identify environmental samplescontaminated with neurotoxic amino acids or neurotoxic derivativesthereof associated with neurological disorders. In one embodiment, thepresent invention provides methods for determining BMAA levels inenvironmental samples including water samples or food items.

In accordance with yet another aspect, the present invention providesmethods for inhibiting neurological disorders in a subject by reducinglevels of a neurotoxic amino acid or embodiment, a subject is a human.In another embodiment, a subject is a human suffering from one or moreneurological disorders. In another embodiment, a subject is a human whois asymptomatic for one or more neurological disorders. In anotherembodiment, a subject is a human who has been identified as being atrisk for developing a neurological disorder. In yet another embodiment asubject is a human who is known or suspected of having been exposed toat least one neurotoxic amino acid or neurotoxic derivative thereofassociated with neurological disorders.

In accordance with one aspect of the present invention, methods areprovided for analyzing tissue samples from a subject, or environmentalsamples used in environmental screening, for one or more forms ofneurotoxic amino acids or neurotoxic derivatives thereof associated withneurological disorders. Methods include analysis of free (e.g., unbound,cytosolic, circulating) forms of neurotoxic amino acids or neurotoxicderivatives thereof associated with neurological disorders,protein-bound forms of neurotoxic amino acids or neurotoxic derivativesthereof associated with neurological disorders (e.g., bound to proteinsor incorporated into proteins), or conjugated forms of neurotoxic aminoacids or neurotoxic derivatives thereof associated with neurologicaldisorders (e.g., conjugated to sugars or lipids). One of skill in theart can determine what forms of neurotoxic amino acid or neurotoxicderivative thereof are present in a sample, and can further determinewhich forms are of diagnostic or predictive interest for a givenembodiment. In one embodiment, tissue samples are analyzed for one ormore forms of BMAA. BMAA can exist in a free (unbound)form in a tissue,or can exist in a protein-bound form, where it may be incorporated intoa protein or it may be otherwise associated with a protein. In oneembodiment, both free and protein-bound BMAA levels are determined. Inanother embodiment, only free BMAA levels are determined. In anotherembodiment, only levels of protein-bound BMAA are determined.

In accordance with another aspect, methods of the invention can bepracticed using any tissue sample obtained from a subject, provided thetissue sample can be analyzed to determine the presence of a neurotoxicamino acid or neurotoxic derivative thereof associated with aneurological disorder. In one embodiment, a tissue sample may beanalyzed to determine the presence of BMAA and if BMAA is present, todetermine the amount of BMAA. Amounts of free BMAA and/or protein-boundBMAA may be quantified, according to the nature of the tissue sample andthe question to be answered in a particular embodiment. In someembodiments, it may be desirable to determine both free andprotein-bound BMAA levels. In other embodiments, it may be desirable todetermine only free BMAA levels. In other embodiments, it may bedesirable to determine only protein-bound BMAA levels. Tissue samplesmay be obtained from a living subject, or may be obtained from apreserved specimen, including stored tissue, biopsy and/or autopsysamples, or museum specimens. Stored tissue may be frozen tissue,histological specimens, tissue dried on solid storage media, or otherforms of stored tissue. Suitable tissue samples include but are notlimited to neurological tissue or non-neurological tissue. Neurologicaltissue can be associated with the central nervous system (CNS),including brain tissue or cerebral-spinal fluid (CSF), or may beassociated with the peripheral nervous system (PNS). Non-neurologicaltissue can be keratinous tissue including but not limited to, hair,skin, nail, including fingernail or toenail, feather, claw, hoof, orhorn. Non-neurological tissue can be non-keratinous tissue including butnot limited to, bood, serum, saliva, or urine. In one embodiment, hairsamples are analyzed to determine the level of protein-bound BMAA. Inanother embodiment, skin is analyzed to determine BMAA levels. In oneembodiment, skin is analyzed to determine free BMAA levels andprotein-bound BMAA. In another embodiment, skin is analyzed to determineonly free BMAA levels. In another embodiment, skin is analyzed todetermine only protein-bound BMAA levels. In yet another embodimentbrain tissue is analyzed to determine BMAA levels. In yet anotherembodiment, samples of cerebrospinal fluid (CSF) are analyzed todetermine the BMAA levels. Brain or CSF tissue may be analyzed todetermine the levels of protein-bound BMAA, free BMAA, or bothprotein-bound and free BMAA, wherein protein-bound BMAA may be bound toneuroproteins or to other proteins.

Screening for Neurological Disorders

The present invention provides screening methods for neurologicaldisorders. As provided herein, neurological disorders (also known asneurologic disorders, or neurologic diseases, or neurological diseases)are disorders that involve the central nervous system (brain, brainstemand cerebellum), the peripheral nervous system (including cranialnerves), and the autonomic nervous system (parts of which are located inboth central and peripheral nervous system). It is understood thatneurological disorders may have complex etiologies, such that one ormore environmental or genetic factors may contribute to development of aneurological disorder in a subject. Neurological disorders includewell-characterized disorders or syndromes such as Alzheimer's disease orParkinson's disease, or may be signs (e.g., aphasia) or symptoms (e.g.,tremors) that are observed in multiple disorders. It is furtherunderstood that the development of a neurological disorder in a subjectmay be due to one factor or a combination of factors. Likewise, it isunderstood that a particular neurological disorder in a subject may bedue to different factors or different combinations of factors thatresulted in the same neurological disorder in other subjects. Screeningmethods as provided herein are suitable for screening for neurologicaldisorders wherein one or more environmental or genetic factor may play apart.

Screening methods include but are not limited to, methods for diagnosingone or more neurological disorders in a subject, methods for predictingthe likelihood of developing one or more neurological disorders in asubject, methods for predicting the severity of a neurological disorderin a subject, and methods for determining exposure of a subject toneurotoxic amino acids or neurotoxic derivatives thereof associated withdeveloping neurological disorders. Methods of the present inventioninclude methods for carrying out repeated testing to generate timeseries data on the presence and levels of neurotoxic amino acids orneurotoxic derivatives thereof in a subject, and/or the presence andlevels of neurotoxic amino acids or neurotoxic derivatives thereof inenvironmental samples.

In accordance with one aspect, methods are provided for diagnosing oneor more neurological disorders in a subject. Methods include correlatingthe presence or absence of a neurotoxic amino acid or neurotoxicderivative thereof in tissue samples from a subject, with other physicalor psychological determinations relevant to assessing neurologicaldisorders. Methods further include correlating the levels of aneurotoxic amino acid or neurotoxic derivative thereof measured in oneor more tissue samples from a subject, with other physical orpsychological determinations relevant to assessing neurologicaldisorders. In one embodiment, tissue samples are obtained from a subjectdiagnosed as having a neurological disorder, BMAA levels are determined,and these results are compared with other physical or psychologicalmeasurements of the subject, as part of a method for diagnosing one ormore neurological disorders. Methods of invention can likewise bepracticed to refine or confirm a diagnosis of one or more neurologicaldisorders, or to exclude other possible diagnoses.

In one embodiment, tissue samples are obtained from a subject suspectedof having a neurological disorder, BMAA levels are determined, and theseresults are compared with other physical or psychological measurementsof the subject, as part of a method for diagnosing one or moreneurological disorders. As disclosed in the Example 4 and Table 3 below,elevated levels of BMAA were found in brain tissue of six Chamorrossuffering from ALS-PDC (lytico-bodig) at the time of their death. Asfurther disclosed in Example 4, elevated levels of BMAA were also foundin brain tissue of Canadian patients diagnosed as suffering fromAlzheimer's Disease (AD) at the time of death.

In yet another embodiment, BMAA levels are measured in tissue samplesfrom a subject who is currently asymptomatic for one or moreneurological disorders. As disclosed in Example 4 and Table 3 below,elevated levels of BMAA were found in brain tissue of a Chamorro patientwho was asymptomatic for ALS-PDC at the time of death. In a furtherembodiment, BMAA levels are measured in tissue samples from a subjectwho is currently asymptomatic for one or more neurological disorders, aspart of a method for identifying subjects at risk of developing aneurological disorder, who may be in need of additional monitoring.

In accordance with another aspect, methods are provided for determiningthe severity of one or more neurological disorders in a subject. Withoutwishing to be limited by this theory, one indicator of the severity of aneurological disorder is the level of a neurotoxic amino acid orneurotoxic derivative thereof measured in a tissue sample from asubject. In one embodiment, the BMAA levels are measured in a tissuesample from a subject diagnosed as having, or suspected of having, oneor more neurological disorders, where higher BMAA levels are correlatewith a more severe neurological disorder.

In accordance with another aspect, methods are provided for predictingthe likelihood of developing a neurological disease. Methods includecorrelating the levels of a neurotoxic amino acid or neurotoxicderivative thereof measured in one or more tissue samples, with otherphysical or psychological determinations relevant to assessingneurological disorders. Methods of the present invention further includecorrelating the levels of a neurotoxic amino acid or neurotoxicderivative thereof measured in one or more tissue samples from asubject, with genetic analysis of the subject to determine thelikelihood of developing a neurological disease. Genetic analysisincludes analysis of family history and/or genotyping tissue samples, aspart of a method for determining the likelihood of developing aneurological disease. Without wishing to be limited by this theory, thelikelihood of a subject developing a neurological disorder shows adirect correlation with the presence of neurotoxic amino acid orneurotoxic derivative thereof measured in a tissue sample from asubject. As disclosed in the Example 4 below, elevated levels of BMAAwere found in brain tissue of six Chamorros suffering from ALS-PDC(lytico-bodig). As further disclosed in Example 4, elevated levels ofBMAA were also found in brain tissue of individuals who died fromAlzheimer's Disease. Accordingly, in one embodiment, BMAA levels aredetermined in tissue samples from subjects having symptoms of one ormore neurological disorders. In another embodiment, BMAA levels aredetermined in tissue samples from subjects asymptomatic for neurologicaldisorders.

In accordance with another aspect, methods are provided for predictingthe severity of a neurological disease in a subject considered to be atrisk for developing one or more neurological disorders. Without wishingto be limited by this theory, levels of BMAA in a tissue sample from asubject are understood to correlate directly with the severity of aneurological disorder once it develops in the subject. Methods of theinvention therefore include correlating the levels of a neurotoxic aminoacid or neurotoxic derivative thereof measured in one or more tissuesamples, with other physical or psychological determinations relevant topredicting the severity of a neurological disorder. Methods of thepresent invention further include correlating the levels of a neurotoxicamino acid or neurotoxic derivative thereof measured in one or moretissue samples from a subject, with genetic analysis of the subject, topredict the severity of a neurological disease in a subject consideredlikely to develop one or more neurological disorders. Genetic analysisincludes analysis of family history and/or genotyping tissue samples.

In accordance with another aspect, methods are provided for longitudinalstudies of neurological disorders by taking tissue samples at repeatedintervals over a period of time and BMAA levels are determined in eachtissue sample, providing time series data on BMAA levels useful forlongitudinal studies. BMAA levels were measured over time in a subjectsuffering from progressive supranuclear palsy (PSP). In yet anotherembodiment, BMAA levels in tissue samples from a subject are repeatedlymeasured over a period of time, in order to determine the level of BMAArelease over time, providing data useful for predicting the likelihoodand/or timing and/or severity of future onset of one or moreneurological disorders.

The invention provides methods for screening neurological disordersincluding but not limited to, Parkinson's disease (PD), Alzheimer'sdisease (AD), progressive supranuclear palsy (PSP), amyotropic lateralsclerosis (ALS), and the neuropathological disease known as ALS-PDC (or,lytico-bodig disease). The teachings of the present disclosure providesufficient guidance to identify other neurological disorders for whichthe present invention provides screening methods: one of skill in theart can practice the methods of the present invention to determine thelevels of a neurotoxic amino acid or neurotoxic derivative thereof intissue samples from a subject, then compare these levels with otherindicia of neurological disease in the subject, and ascertain whether acorrelation exists between levels of the neurotoxic amino acid orneurotoxic derivative thereof, and indicia of a particular neurologicaldisease.

In accordance with one aspect, methods as provided herein are suitablefor screening for neurodegenerative disorders with neurofibrillarytangles (known as neurofibrillary tangle disorders or NFT disorders),including but not limited to argyrophilic grain disease, Alzheimer'sdisease, ALS-PDC of Guam, corticobasal degeneration, mytonic dystrophy,Pick's disease, postencephalitic parkinsonism, primary progressiveaphasia, progressive supranuclear palsy (PSP), and subacute sclerosispanencephalitis. These disorders are generally characterized byneurofibrillary degeneration (NFD) leading to intraneuronal accumulationof pathological tau proteins into abnormal filaments and sometimescalled “tauopathies.” Different NFT disorders have distinct taupathologies (that is, tau protein isoforms and distribution in brain).In accordance with one aspect of the invention, levels of a neurotoxicamino acid or neurotoxic derivative thereof, e.g., BMAA, are measured ina tissue sample from a subject known or suspected to be suffering froman NFT disorder. In accordance with another aspect, levels of neurotoxicamino acid or neurotoxic derivative thereof, e.g., BMAA, are measured ina tissue sample from a subject who is asymptomatic for an NFT disorder.In accordance with another aspect, levels of modified acids, e.g., BMAA,are measured in a tissue sample from a subject who is asymptomatic foran NFT disorder but is considered to be at risk for developing an NFTdisorder, e.g., based a family history of NFT disorders, or based onknown or suspected exposure to environmental factors associated with NFTdisorders. Analysis of brain tissue according to the methods of thepresent invention permits comparison of BMAA levels with other factorsincluding, but not limited to, identifying whether NFTs are present,identifying which tau protein isoforms are present, and investigatingthe distribution pattern of tau protein and/or NFTs in the brain of thesubject.

In one embodiment, BMAA levels are measured in brain tissue of a subjectknown or suspected to be suffering from ALS-PDC, which has a distincttau pathology from other NFT disorders. In another embodiment, BMAAlevels are measured in brain tissue of a subject known or suspected tobe suffering from Alzheimer's disease, which has a distinct taupathology from other NFT disorders. In another embodiment, BMAA levelsare measured in brain tissue of a subject known or suspected to besuffering from progressive supranuclear palsy (PSP) and/or corticobasaldegeneration, which have a distinct tau pathology from other NFTdisorders. In another embodiment, BMAA levels are measured in braintissue of a subject known or suspected to be suffering from Pick'sdisease, which has a distinct tau pathology from other NFT disorders. Inanother embodiment, BMAA levels are measured in brain tissue of asubject known or suspected to be suffering from myotonic dystrophy,which has a distinct tau pathology from other NFT.

In one embodiment, BMAA levels in tissue samples from subjects diagnosedas suffering from Alzheimer's disease are determined according themethods of the present invention. In another embodiment, BMAA levels aredetermined in tissue samples from subjects who are asymptomatic forAlzheimer's disease. In yet another embodiment, BMAA levels in tissuesamples from subjects who are asymptomatic for Alzheimer's, but aresuspected to be at risk of developing Alzheimer's disease, aredetermined according to the methods of the present invention.

In accordance with another aspect, methods as provided herein are usefulfor distinguishing between neurological disorders and/or screeningindividuals having a neurological disorder for additional neurologicaldisorders. In one embodiment, an individual with Down's syndrome, aneurological disorder caused by trisomy of chromosome 21 andcharacterized by symptoms including NFTs, is screened for neurotoxicamino acid or neurotoxic derivative thereof as provided herein. In oneembodiment, detecting the presence of BMAA in a subject suffering fromDown's syndrome can be used to identify a subject at risk of developinga neurological disorder associated with a neurotoxic amino acid orneurotoxic derivative thereof. In another embodiment, detecting thepresence of BMAA in a subject suffering from Down's syndrome can be usedto distinguish between multiple neurological disorders in a subject. Inanother embodiment, detecting the presence of BMAA in a subjectsuffering from Down's syndrome can be used to distinguish betweenpossible causes (etiologies) of a sign or symptom of a neurologicaldisorder.

In accordance with another aspect, methods as provided herein are usefulfor screening for dementias including but not limited to Alzheimer'sdisease (AD), Lewy body dementia (LBD, also called dementia with Lewybodies (DLB)) and vascular dementia. In accordance with another aspect,methods as provided herein are useful for screening for movementdisorders including but not limited to Parkinson's disease (PD),dystonias (sustained involuntary muscle contractions), Huntington'sdisease (Huntington's chorea), multiple system atrophy, progressivesupranuclear palsy, corticobasal degeneration, dyskinesias, essentialtremor, hereditary spastic paraplegia, myoclonus, restless legssyndrome, Rett syndrome, spasticity, Sydenham's chorea, Tourette'ssyndrome, and Wilson's disease. In accordance with another aspect,methods as provided herein are useful for screening for motor neurondiseases (MND) including but not limited to amyotrophic lateralsclerosis (ALS), progressive muscular atrophy (muscular dystrophy (MD)),and postpolio syndrome. In accordance with yet another aspect, methodsas provided herein are useful for screening for amyotrophic lateralsclerosis/parkinsonism-dementia complex of Guam (ALS/PDC, also known aslytico-bodig).

It is understood that methods as provided herein are suitable forscreening for neurological disorders regardless of whether any signs orsymptoms of neurological disorders are present. As disclosed in theExample 4 below, elevated levels of BMAA were found in brain tissue fromone Chamorro who was asymptomatic for ALS-PCD, while anotherasymptomatic Chamorro did not have detectable BMAA levels. This resultis consistent with the observation that neurofibrillary tangles havebeen observed in brain tissue of certain Chamorros who did not showsymptoms of ALS-PDC.

It is further understood that methods as provided herein are suitablefor screening for neurological disorders regardless of whether aparticular neurological disorder can be diagnosed. Because distinctdisorders often share similar signs and symptoms (e.g., tremors,dementia, aphasia), methods of the present invention may be suitable aspart of an initial screening for neurological disease, wherein theresults of the initial screening are relied upon for determining whatfurther tests are needed for a thorough assessment. For example,subjects with ALS-PDC can have symptoms similar to Alzheimer's diseaseor Parkinson's disease, or both diseases, and although ALS-PDC isconsidered a separate disorder, it is also possible for a subject withALS-PDC to also suffer from Alzheimer's disease or Parkinson's disease.Accordingly, measurement of BMAA levels in a subject may aid inidentifying which neurological disorders are present are contributing tothe signs and symptoms observed in the subject.

Neurological disorders include, but are not limited to: acquiredepileptiform aphasia; acute disseminated encephalomyelitis;adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardisyndrome; Alexander disease; Alpers' disease; alternating hemiplegia;Alzheimer's disease (AD); amyotrophic lateral sclerosis (ALS);amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam(ALS/PDC); anencephaly; Angelman syndrome; angiomatosis; anoxia;aphasia; apraxia; arachnoid cysts; arachnoiditis; Amold-Chiarimalformation; arteriovenous malformation; Asperger syndrome; ataxiatelangiectasia; attention deficit hyperactivity disorder; autism;autonomic dysfunction; Batten disease; Behcet's disease; Bell's palsy;benign essential blepharospasm; benign focal amyotrophy; benignintracranial hypertension; Binswanger's disease; blepharospasm;Bloch-Sulzberger syndrome; brachial plexus injury; brain abscess; braininjury; brain tumor; spinal tumor; Brown-Sequard syndrome; Canavandisease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome;central pontine myelinolysis; cephalic disorder; cerebral aneurysm;cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism;cerebral palsy; Charcot-Marie-Tooth disease; Chiari malformation;chorea; chronic inflammatory demyelinating polyneuropathy (CIDP);chronic pain, chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirusinfection; dancing eyes-dancing feet syndrome; Dandy-Walker syndrome;Dawson disease; De Morsier's syndrome; Dejerine-Klumpke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essentialtremor; Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giantcell inclusion disease; globoid cell leukodystrophy; Guillain-Barresyndrome; HTLV-1 associated myelopathy; Hallervorden-Spatz disease; headinjury; headache; hemifacial spasm; hereditary spastic paraplegia;heredopathia atactica polyneuritiformis; Herpes zoster oticus; Herpeszoster; Hirayama syndrome; holoprosencephaly; Huntington's disease;hydranencephaly; hydrocephalus; hypercortisolism; hypoxia;immune-mediated encephalomyelitis; inclusion body myositis;incontinentia pigmenti; infantile phytanic acid storage disease;infantile Refsum disease; infantile spasms; inflammatory myopathy;intracranial cyst; intracranial hypertension; Joubert syndrome;Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome; KlippelFeil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Laforadisease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome;lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh'sdisease; Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy;Lewy body dementia; lissencephaly; locked-in syndrome; Lou Gehrig'sdisease (ALS); lumbar disc disease; Lyme disease, neurological sequelae;Lytico-Bodig syndrome (ALS-PCD); Machado-Joseph disease; macrencephaly;megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease,meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly;migraine; Miller Fisher syndrome; mini-strokes; mitochondrialmyopathies; Mobius syndrome; monomelic amyotrophy; motor neuronedisease; Moyamoya disease; mucopolysaccharidoses; multi-infarctdementia; multifocal motor neuropathy; multiple sclerosis; multiplesystem atrophy with postural hypotension; muscular dystrophy; myastheniagravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy ofinfants; myoclonus; myopathy; myotonia congenita; narcolepsy;neurofibromatosis; neuroleptic malignant syndrome; neurologicalmanifestations of AIDS; neurological sequelae of lupus; neurologicalsequelae of Lyme disease; neuromyotonia; neuronal ceroid lipofuscinosis;neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeodsyndrome, occipital neuralgia; occult spinal dysraphism sequence;Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus;optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia;Parkinson's disease (PD); paramyotonia congenita; paraneoplasticdiseases; paroxysmal attacks; Parry Romberg syndrome;Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy;persistent vegetative state; pervasive developmental disorders; photicsneeze reflex; phytanic acid storage disease; Pick's disease; pinchednerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive sclerosing poliodystrophy; progressivesupranuclear palsy (PSP); pseudotumor cerebri; Ramsay-Hunt syndrome;Ramsay Hunt syndrome Type I; Ramsay Hunt syndrome Type II; Rasmussen'sEncephalitis; reflex sympathetic dystrophy syndrome; Refsumdisease—infantile; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; SaintVitus Dance; Sandhoff disease; Schilder's disease; schizencephaly;septo-optic dysplasia; shingles; Shy-Drager syndrome; Sjogren'ssyndrome; Soto's syndrome; spasticity; spina bifida; spinal cord injury;spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome;stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis;subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope;syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporalarteritis; tethered spinal cord syndrome; Thomsen disease; thoracicoutlet syndrome; tic douloureux; Todd's paralysis; Tourette's syndrome;transient ischemic attack; transmissible spongiform encephalopathies;transverse myelitis; traumatic brain injury; tremor; trigeminalneuralgia; tropical spastic paraparesis; tuberous sclerosis; vasculitisincluding temporal arteritis; Von Hippel-Lindau Disease (VHL);Wallenberg's syndrome; Werdnig-Hoffinan disease; West syndrome; Williamssyndrome; Wilson's disease; Zellweger syndrome.

Screening for Environmental Factors Associated with NeurologicalDisorders.

In accordance with one aspect, methods are provided for screening forenvironmental factors associated with neurological disorders.Environmental factors associated with neurological disorders include,but are not limited to, a neurotoxic amino acid or neurotoxic derivativethereof, e.g., BMAA. Screening as provided herein includes, but is notlimited to, testing environmental samples to determine actual orpotential exposure of a subject to a neurotoxic amino acid or neurotoxicderivative thereof associated with neurological disorders. Anenvironmental sample may be obtained from material that is ingested,e.g. a water sample or a food sample. An environmental sample may bematerial that is deliberately ingested, e.g., water used for drinking,or plants or animals that are part of the food supply or food chain.Alternately, an environmental sample may be obtained from material thatis incidentally ingested, e.g., material from an organism whose contentsor secretions become associated with other ingested material, such ascyanobacterial symbionts present in plants used for food, orcyanobacteria in water used for washing or drinking.

In one embodiment, the BMAA levels in environmental samples are measuredto determine the actual of potential exposure of a subject to BMAA.Measurements of BMAA levels in environmental samples leads to adetermination of potential or actual exposure to BMAA, and thesemeasurements can be used to predict the likelihood that neurologicaldisorders will develop in a subject exposed to these environmentalsamples. It is understood that BMAA in cycad tissues and other planttissues, is produced by cyanobacterial symbionts and taken up by thecycads and other organisms that feed on cycads (Example 3). Numeroussamples from an archive of cyanobacteria have been tested for theability to produce BMAA, and nearly all strains tested produce BMAA. Inlight of the discovery of symbiotic cyanobacteria as the source of BMAAin cycads (Example 3), coupled with the near ubiquity of cyanobacteriain soil and water, and the discovery that many cyanobacterial strainsproduce BMAA, it is proposed that BMAA may be present in manyenvironments. Accordingly, methods of the present invention may furtherinclude screening environmental samples for the presence ofcyanobacteria in addition to screening for particular factors such asBMAA.

In accordance with another aspect, an environmental sample is waterknown to contain cyanobacteria. In another embodiment, an environmentalsample is water suspected of containing cyanobacteria. In anotherembodiment, an environmental sample is water whose contents are unknown.In another embodiment, an environmental sample may be an food animalthat ingests cyanobacteria-containing water, e.g., a fish, bird, deer,or domesticated animal. In another embodiment, an environmental samplemay be lichen or moss or liverworts that contain or live in symbiosiswith cyanobacteria.

In another embodiment, an environmental sample may be a marine orfreshwater alga or a marine or freshwater fungus that contain or live insymbiosis with cyanobacteria. In another embodiment, an environmentalsample may be a marine or freshwater invertbrate that contains or livesin symbiosis with cyanobacteria. In another embodiment, an environmentalsample may be stromatolite, or a petrochemical deposit, or a mineraldeposit left by cyanobacteria. In another embodiment, an environmentalsample may be a food animal that ingests a plant, lichen, moss, alga,marine invertebrate, that contain cyanobacteria or a stromatolite,petrochemical deposit, or mineral deposit left by cyanobacteria, e.g. areindeer, caribou, deer, moose, marine or freshwater fish, bird,reptile, or domesticated animal.

In accordance with another aspect, an environmental sample is screenedto determine if the sample is associated with a neurological disorder,by detecting the presence of cyanobacteria that produce a neurotoxicamino acid or neurotoxic derivative thereof, in the environmentalsample. By screening environmental samples to detect cyanobacteria thatproduct neurotoxic amino acids or neurotoxic derivatives thereof, it ispossible to determine actual or potential exposure of a subject toenvironmental factors associated with a neurological disorder.Neurotoxic amino acids or neurotoxic derivatives thereof, e.g., BMAA,have been found in many cyanobacteria strains of genera including, butnot limited to, Nostoc and Anabena.

In accordance with another aspect, a plurality of environmental samplesis tested to determine the levels of neurotoxic amino acids orneurotoxic derivatives thereof associated with neurological disorders,at different levels throughout a food chain. Without wishing to belimited by this theory, biomagnification of factors associated withneurological disorders, e.g., BMAA, can occur by accumulation of afactor in tissues of organisms at different trophic levels, with theresult that consumption of an organism from a higher trophic level maygive a much higher exposure to a neurotoxin than consumption of anorganism from a lower trophic level. In one embodiment, a plurality ofenvironmental samples is tested in a food chain, including cycadcoralloid roots, cycad leaves, cycad seeds, and tissue samples fromflying foxes (bats) known to eat cycad seeds. In another embodiment, aplurality of environmental samples is tested in a food chain, includingwater, aquatic plants, food animals that ingest the water or aquaticplants, e.g., fish birds, a wild or domesticated animal, and carnivoresthat ingest plant-eating animals. In one embodiment, a plurality ofenvironmental samples can be tested to determine whether a factor suchas BMAA is found in a particular food chain. After testing a pluralityof environmental samples, levels of a neurotoxic amino acid orneurotoxic derivative thereof can be compared and analyzed for evidenceof accumulation or biomagnification in the food chain.

In accordance with a further aspect, a tissue sample from a subject isalso tested, in addition to testing environmental samples for aneurotoxic amino acid or neurotoxic derivative thereof associated withneurological diseases. This provides methods for determiningaccumulation or biomagnification of environmental factors (neurotoxicamino acids or neurotoxic derivatives thereof) in a food chain andcorrelating levels of these environmental factors in each step of thefood chain with the frequency or severity of neurological disorders insubjects that consume material from various trophic levels of the foodchain. In one embodiment, a tissue sample from a subject with symptomsof, or a diagnosis of, a neurological disorder is tested for aneurotoxic amino acid or neurotoxic derivative thereof associated withneurological diseases. In another embodiment, a tissue sample from asubject asymptomatic for a neurological disorder is tested for aneurotoxic amino acid or neurotoxic derivative thereof associated withneurological diseases. This aspect of the present invention provides apowerful tool for linking neurological disorders with exposure toenvironmental factors that are known or suspected to be associated withneurological disorders. As shown in Example 4 below, elevated BMAAlevels were detected in brain tissues of subjects who died of ALS-PDCafter known exposure to food sources that were known or suspected tocontain BMAA—that is, the subjects who died of ALS-PDC were Chamorroswho had eaten a traditional Chamorro diet at some time in their life.Without wishing to be limited by this theory, these results arecongruent with the results presented in Example 2 below, showing highconcentrations of BMAA in specimens of flying foxes, a traditionalChamorro food, leading to the prediction by the inventors thatconsumption of a single flying fox would result in a dose of BMAAequivalent to the dose obtained by eating 174-1,014 kg of processedcycad flour. In addition, elevated BMAA levels were detected in oneChamorro subject who was asymptomatic for ALS-PDC and died of othercauses. Without wishing to be limited by this theory, it should be notedthat this result is congruent with the report by Forman et al. on astudy of 30 Chamorros (Forman et al., 2002, Am J Pathol 160: 1725-1731),which found neurofibrillary tangles in brain tissue of both affected(ALS-PDC) and unaffected (asymptomatic) Chamorros. In contrast, anotherChamorro subject who was asymptomatic for ALS-PDC and died of othercauses, did not have detectable BMAA levels in brain tissue.

Another aspect of the invention provides methods for detectingenvironmental contamination by environmental factors associated withneurological disorders. Surprisingly, elevated BMAA levels were found inbrain tissue of non-Chamorro (Canadian) subjects who had suffered fromAlzheimers disease (see, Examples below) and in a non-Chamorro(Canadian) suffering from progressive supranuclear palsy (PSP). Inaccordance with this aspect of the invention, elevated BMAA in braintissue of these Alzheimer's disease patients, and the elevated BMAA intissue samples from a PSP patient, indicated that these subjects hadbeen exposed to environmental sources of BMAA at some time in theirlife. These results suggested that bioaccumulation of cyanobacterialBMAA may occur through different food chains in other areas. Since thefrequency of illness in a population exposed to neurotoxins is afunction of dose, even low levels of progressive neurological disordersmight be related to exposure to low concentrations of BMAA in watersupplies contaminated by cyanobacteria. Accordingly, environmentalscreening as provided herein can be carried out to investigate possibleenvironmental sources of BMAA or other environmental factors associatedwith neurological disorders. Environmental screening as provided hereincan be carried out to prevent or minimize exposure of other subjects toBMAA or other environmental factors associated with neurologicaldisorders, thereby decreasing the risk of developing a neurologicaldisorder associated with BMAA or other factors.

In accordance with a further aspect, the present invention can be usedto protect subjects from exposure to environmental factors associatedwith neurological disorders by developing assays and assay kits for suchfactors. In one embodiment, assays are provided to test food samples,including plant or animal matter, for BMAA. In another embodiment,assays are provided to test water supplies for BMAA. In yet anotherembodiment, assays kits are provided for environmental screening forBMAA, where kits include materials for practicing methods of theinvention to test water supplies, food supplies, and other environmentalsamples, to protect subjects from exposure to BMAA. In accordance withanother aspect, assays and assay kits for BMAA can be used for publichealth purposes, e.g., to indicate contamination of a water supply orfood source with cyanobacteria that produce BMAA.

Reservoirs of Neurotoxic Amino Acids or Neurotoxic Derivatives thereofAssociated with Neurological Disorders

Neurotoxic amino acids or neurotoxic derivatives thereof may accumulatein one or more endogenous reservoirs in a subject. BMAA is of naturalorigin, unlike certain other environmental factors associated withneurological disorders, e.g., mercury or PCBs. Protein-bound BMAA hasbeen found in various tissues, suggesting possible incorporation duringprotein synthesis or through association with a carrier protein. Earlierreports indicated that 90% of injected BMAA is not eliminated fromeither urine or feces in rats, suggesting that BMAA accumulates insubjects, particularly in mammals. These findings, in combination withthe epidemiological observation of a period of latency associated withALS-PDC, suggest an endogenous neurotoxic reservoir from which BMAA maybe released over time, probably as a result of protein metabolism.Without wishing to be bound by this theory, the BMAA reservoir mayfunction as a “slow toxin” causing damage in a subject through at leastfive different possible neuropathological routes: (1) incorporation ofnon-protein amino acids such as BMAA may alter tertiary folding ofneuroproteins, altering their biological activity; (2)protein-associated BMAA may form dimers that covalently bind metal ions,which could result in a protein punctuated with reactive non-proteinamino acid complexes that alter ionic balance in neuronal cells,generate free radicals, or even catalyze deleterious chemical processes;(3) capture and release of metal ions such as those of Zn²⁺, Cu²⁺, orCa²⁺ by BMAA complexes may interfere with the proper function of NMDAand AMPA receptors; (4) BMAA incorporation may truncate proteins beforecompleted synthesis or collapse proteins after release from theribosome, where such truncation of protein synthesis is characteristicof many of the tauopathies (NFT disorders); and (5) BMAA may be slowlyreleased in free form through protein metabolism in the brain, servingas an agonist at AMPA, NMDA, and other neuroreceptors. The latteractivity may effectively translate a single ingestion, or episodicingestions, of BMAA into a highly prolonged, constant low level exposureof BMAA within the superior frontal gyrus, possibly resulting in neurondeath via excitotoxicity. Etiologically, such prolonged low-levelexposure may not produce acute disease, such as has been observed inanimal models, but instead might result in both the latency andprogressive nature typified by ALS-PDC among the Chamorro people.Protein-associated BMAA in endogenous reservoirs may therefore be thehypothesized “slow toxin” of ALS-PDC.

A study was carried out to determine whether BMAA is associated withproteins in the food chain. As shown in the Examples below,protein-bound BMAA was measured by removing all free amino acids fromsamples of cyanobacteria, cycad seed tissue, flying fox (bat) hair andskin, and human brain tissue. After all free amino acids were removed,the protein fraction was hydrolyzed. The hydrolyzed proteins were thentested for BMAA. Protein-bound BMAA was found in all tissues tested.Without wishing to be limited by this theory, this finding ofprotein-bound BMAA in all tissues suggests possible incorporation duringprotein synthesis, or through association with a carrier protein.

The results disclosed herein indicate that BMAA, which originates withcyanobacteria, accumulates in plant and animal tissues that become partof the food chain. In particular, these results shows that BMAA ofcyanobacterial original accumulates in the Guam food chain, where it isbiomagnified by flying foxes who consume BMAA-containing cycad seeds andaccumulate BMAA, and may be further biomagnified when Chamorro peopleeat flying foxes containing large amounts of BMAA, with the result thatBMAA accumulation in brain tissue is associated with the ALS-PDCneurological disorder among the Chamorro people.

The brain tissue in which BMAA was detected exhibited intercellularneurofibrillary tangles, extracellular neurofibrillary tangles and cellloss. In one Lytico-Bodig (ALS-PDC) patient, no unbound BMAA was foundin the brain tissue, but more than 1 mg/g BMAA was recovered from theprotein-bound fraction. In all other patients, there was roughly a60-130 fold greater quantity of protein-bound BMAA compared to the BMAArecovered from the free amino acid pool (free BMAA). This suggests thatthe rate of amino acid flux between the protein-bound BMAA and freeBMAA, varies between individuals, and may be subject to nutritionalstatus, genetic proclivities, age, endocrine function, or idiopathicdifferences. Without wishing to be limited by this theory, protein-boundBMAA represents the BMAA reservoir for a subject, and may be the morerobust indicator in screening for neurological disorders. The relativeamounts of BMAA in the protein-bound form (e.g., in the “endogenousneurotoxic reservoir”) and in the unbound form in the free amino acidpool should be compared with clinical manifestations of neurologicaldisorders, to determine the dose/duration relationship.

The possibility of alternative pathways for bioaccumulation ofcyanobacterial BMAA in other parts of the world is supported by thefinding of protein-associated BMAA in brain tissue of Alzheimer'spatients from Canada. As shown in Table 3, high levels of protein-boundBMAA (149-1190 μg/g) were found in frontal cortex tissue of all sixChamorrow patients who had died from ALS-PDC. Frontal cortex tissue fromfive of six Chamorro patients who had died from ALS-PDC also had highlevels of free BMAA (3-10 μg/g). In addition, significant amounts offree and protein-bound BMAA was found in one asymptomatic Chamorropatient who did not die of ALS-PDC, consistent with previous findings ofChamorros who exhibited no clinical manifestations of ALS-PCD, but whoshowed signficant neuroanatomical pathologies when autopsied.Significant concentrations of BMAA were found in the frontal gyrus ofbrain cortex of two Canadian patients who were diagnosed as sufferingfrom from Alzheimer's disease at the time of their death. In the samestudy, brain tissue of thirteen Canadian patients who did not have adiagnosis of Alzheimer's disease and died of other causes, did not havedetectable levels of BMAA. The unexpected finding of BMAA in Canadianpatients suffering from Alzheimer's disease, which is a disorder with atau pathology distinct from that of ALS-PDC, suggests that BMAA ispresent and may accumulate in other food chains, finally accumulating inhuman subjects in a reservoir from which BMAA may be released over time.Since there was no indication that the Canadian Alzheimer's patientsever lived in Guam or consumed a Chamorro diet, the MDAA found in theirbrains must ultimately be traced to a non-cycad source. Accordingly, thepresent invention provides methods for determining exposure andbiomagnification of environmental factors associated with neurologicaldisorders, including methods for identifying the vectors ofbiomagnification.

Inhibiting Neurological Disorders: Treatment and/or Prevention

In accordance with yet another aspect, the present invention providesmethods for inhibiting neurological disorders in a subject. Inaccordance with one aspect, neurological disorders are inhibited byreducing levels of a neurotoxic amino acid or neurotoxic derivativethereof associated with neurological disorders. In accordance withanother aspect, neurological disorders are inhibited by reducing thetoxic effect of a neurotoxic amino acid or neurotoxic derivative thereofassociated with neurological disorders. In accordance with one aspect,neurological disorders are inhibited by interfering with the interactionof a neurotoxic amino acid or neurotoxic derivative thereof with targetmolecules. It is understood that neurological disorders can be inhibitedby treating one or more existing disorders, or by treating earlysymptoms or signs of disorders, or by preventing the onset of one ormore disorders, or by preventing the progression (worsening) of one ormore disorders.

In accordance with one aspect, levels of a neurotoxic amino acid orneurotoxic derivative thereof can be reduced by releasing the neurotoxicamino acid or neurotoxic derivative thereof from an endogenous reservoirin the subject. The invention further provides methods for minimizingdamage from releasing a neurotoxic amino acid or neurotoxic derivativethereof from endogenous reservoirs including, but not limited to,providing neuroprotectant compounds that interfere with the interactionof the neurotoxic amino acid or neurotoxic derivative thereof with atarget molecule, or providing compounds to bind and inactivate theneurotoxic amino acid or neurotoxic derivative. In one embodiment, aneurological disorder can be inhibited by releasing (“draining”) BMAAfrom endogenous reservoirs, to prevent accumulation in a subject. In oneembodiment, a subject is infused with a monoclonal antibody against BMAAwhen BMAA is released from the reservoir. In another embodiment, thesubject is infused with glutamate as a neuroprotectant compound whenBMAA is released from the reservoir. In a further embodiment, a subjectis infused with metal chelating compounds to absorb metal ions releasedwhen BMAA is released from the reservoir.

In accordance with another aspect, the toxic effects of a neurotoxicamino acid or neurotoxic derivative thereof associated with aneurological disorder are reduced by adding neuroprotectant compoundsthat interfere with the interaction of the neurotoxic amino acid orneurotoxic derivative thereof with target molecules, thereby dilutingthe effective level of the neurotoxic amino acid or neurotoxicderivative thereof. In one embodiment, the toxic effects of BMAA arereduced by increasing the intracellular levels of glutamic acid(possibly ionized as glutamate) or glutamic acid homologs, such that theeffective pool of BMAA is diluted and target molecules are protected. Inthis embodiment, glutamic acid or glutamate functions as aneuroprotectant compound. In a further embodiment, chelating agents areadded.

Kits for Screening for Neurotoxic Amino Acids or their Derivatives

The present invention further provides kits for screening a subjecthaving or at risk of having a neurological disorder, wherein the kitsinclude a means for obtaining a tissue sample from the subject and ameans for analyzing the tissue sample to determine the presence of aneurotoxic amino acid or neurotoxic derivative thereof associated withthe neurological disorder. Means for obtaining tissue samples are knownin the art. Means for analysing a tissue sample to determine thepresence of a neurotoxic amino acid or neurotoxic derivative thereof areknown in the art; non-limiting embodiments are disclosed herein. The kitmay include means for determining the presence of a glutamate receptoragonist such as a methylated alanine, in particular BMAA. The kit mayinclude means for analyzing protein-bound BMAA, free BMAA, or bothprotein-bound BMAA and free BMAA in the sample. In accordance with oneaspect, kits of the present invention include “control” samples of oneor more neurotoxic amino acids being determined, to facilitate bothdetection and quantification of each neurotoxic amino acid or neurotoxicderivativethereof being determined in the sample. In one embodiment, akit includes thin layer chromatography (TLC) plates, such that tissuesamples and control samples can be spotted on plates, separated bysolvent migration, and determined.

In accordance with one aspect of the invention, a kit may include meansfor analyzing a plurality of tissue samples from the subject. In oneembodiment, the tissue samples may include a sample of a tissue in whicha neurotoxic amino acid or neurotoxic derivative thereof is known toaccumulate and a sample of a tissue in which neurotoxic amino acid orneurotoxic derivative thereof is known to not accumulate, therebypermitting a determination of whether neurotoxic amino acids haveaccumulated in certain tissues. In another embodiment, the tissuesamples may include a sample of at least two distinct tissues in which aneurotoxic amino acid or neurotoxic derivative thereof is known toaccumulate, permitting a determination of the relative levels ofaccumulation in different tissues. In accordance with another aspect ofthe invention, a kit may include means for performing repeated screeningof the subject. In one embodiment, the subject is screened at repeatedintervals that may stretch over days, months, or years. Kits of thepresent invention can be used in longitudinal studies as describedabove.

EXAMPLES Example 1 Distribution of BMAA in Cycas micronesica Hill

The concentrations of BMAA and glutamic acid (GLU) were measured indifferent cycad tissues. Other known nitrogenous neurotoxins, includingthe carbamate precursors DAB, DAP, and ODAP (also known as BOAA) werealso measured.

BMAA, GLU, DAB, DAP, and ODAP (BOAA) were measured in wild seeds ofCycas micronesica Hill collected from Guam, and in various tissues fromliving specimens of Cycas micronesica Hill of known provenance in theFairchild Tropical Gardens, the Montgomery Botanical Center, and theNational Tropical Botanical Garden. Herbarium tissue from the NationalTropical Botanical Garden was also analyzed, as BMAA has been found tobe stable in dried mammal specimens of great age (Banack & Cox, 2003).

BMAA and GLU were quantified from free amino acid extracts of cycadtissues following the techniques of Kisby, Roy & Spencer (1988) withminor modifications. Aqueous or trichloroacetic acid sample extractswere derivatized with 6-aminoquinolyl-N-hydrozysuccinimidyl carbamate(ACQ) following standardized protocols (Cohen & Michaud, 1993). Freeamino acids were separated by reverse phase separation on a gradientHPLC system (Waters 717 Automated Injector, Waters 1525 Binary SolventDelivery System and Waters Nova-Pak C18 column, 300 mm×3.9 mm) at 37° C.Individual compounds were eluted from the column with a gradient elutionof 140 mM sodium acetate, 5.6 mM triethylamine, pH 5.2 and 60%acetonitrile (Cohen & Michaud, 1993). The identities of the BMAA peakand the GLU peak were confirmed by comparison to authenticated standardsand were re-verified by modified gradient elution. The concentration ofBMAA and GLU in samples was determined by fluorescence detection (Waters2487 Dual-1 Fluorescence Detector) with excitation at 250 nm andemission at 395 nm with concurrent UV detection (Waters 2488 UVdetector) at 254 nm. Detection of the ACQ-derivatized BMAA was dependenton concentration and comparison of equal amounts of BMAA and anorleucine internal standard resulting in a mean response of 51.2%.These data may be indicative of internal quenching of the derivative,but did not significantly affect sample quantification as the percentageresponse was consistent across the quantifiable concentration range. Thelimits of detection (LOD) and limits of quantification (LOQ) weredetermined by a concentration gradient of an authenticated standard(Sigma Chemical Co., St. Louis, Mo.). The LOD and the LOQ were 0.00013μmoles and 0.013 μmoles respectively per injection for all analyses. Asshown in Table 1 below, for purposes of data interpretation, all sampleanalyses were quantified within the range of the LOQ, or were reportedas not present. Authenticated standards for DAB (2,4-diamino butyrate),DAP (2,3-diaminopropionate) and BOAA (β-N-oxalyl-amino-L-alanine) werealso used to identify the corresponding HPLC peaks for presence orabsence of these compounds, but no attempt was made to quantify theirconcentrations. Breeze scientific software (Trinity Consultants Inc.,Dallas Tex.) was used to control system operation and collect andanalyze data.

As shown in Table 1, BMAA and GLU occurred in various cycad tissues, asdid compounds corresponding to the neurotoxins DAB, DAP, and BOAA. GLUwas found in substantially higher quantities than BMAA in all tissues.

TABLE 1 BMAA and GLU concentrations (μg/g) in Cycas micronesica Hilltissues. BMAA GLU Sample mean mean Tissue size (μg/g) (μg/g) BOAA DAPDAB root, non-coralloid 3 — 45,922 † root, coralloid, non-infected 2 —86,900 † root, coralloid, mild infection 2 37 9,449 † root, coralloid,heavy infection 2 2 22,988 † † roots, coralloid, senescent infection 1 —6,435 — senescent stem, inner cortex 3 — 22,781 — stem, outer cortex 1 —103,919 — stem, xylem 3 — 18,323 — Leaf 3 13 65,953 † † leaf mucilage 1— — — male sporophyll, immature 1 663 57,334 — male sporophyll, mature 1— 57,819 — male sporangia, immature 2 1546 68,202 † male sporangia,mature 2 11 — † † † female sporophyll 2 — — — † seed sarcotesta 3 9 — —— — seed sarcotesta, outer integument 3 1161 — † — — layer seedgametophyte 3 240 35,700 † — — Samples were analyzed by HPLC andcompared with amino acid standards. Legend: † = trace amounts — = notdetectable

For purposes of comparison, the relative concentrations of BMAA and GLUwere normalized by dividing the concentration for each molecule by themaximum concentration found (FIG. 1). The highest concentrations of BMAAwere found in plant reproductive tissues. Concentrations of GLU weresimilar in all tissues tested, and showed no clear pattern ofdistribution. Although GLU appeared to be distributed throughout theplant without any apparent pattern, BMAA was concentrated in the maleand female reproductive tissue where it may act as a deterrent toherbivory. The high concentration of BMAA in the outer layer of thesarcotesta showed that any animal that forages on cycad sarcotesta(e.g., flying foxes) would be exposed to high cumulative doses of BMAAover time. Other neurotoxic compounds were detected but not quantifiedin various parts of the cycad tissues including BOAA, DAB, DAP (Table1).

Example 2 Biomagnification of Cycad Neurotoxins in Flying Foxes (Bats)of Guam

BMAA levels were measured in tissues of Cycas micronesica Hill fromGuam, and tissues of Pteropus mariannus mariannus, an indigenous flyingfox (bat) of Guam. Because Pteropus mariannus mariannus is now highlyendangered, BMAA levels were measured in skin tissue from museumspecimens of three flying foxes that were collected five decades ago inGuam, preserved as dried study skins, and deposited at the Museum ofVertebrate Zoology (MVZ), at the University of California, Berkeley.

Seeds of Cycas micronesica Hill collected from Guam, and samples ofprocessed (washed, detoxified) cycad flour specimens collected from Guam(Dr. J. C. Steele, 1987-1988) were analyzed for their BMAA content.Traditional preparation of cycad flour by the Chamorros commonlyinvolves soaking the gametophytes of the seeds of Cycas micronesia Hillfor about 3 weeks with changes of water every 2-3 days.

BMAA was detected using high performance liquid chromatography (HPLC),and results were confirmed with thin layer chromatography (TLC) and gaschromatography-mass spectroscopy (GC-MS). For BMAA analysis, free aminoacid extracts of flying fox and cycad tissues were prepared. Tissuesamples were rehydrated for 30 minutes with water or trichloroaceticacid (mean tissue prep 80 mg/ml±32 SD), macerated, and filtered.Extracts were derivatized with 6-aminoquinolyl-N-hydrozysuccinimidylcarbamate (ACQ) following standardized protocols. Free amino acids wereseparated by reverse phase separation on a gradient HPLC system (Waters717 Automated Injector, Waters 1525 Binary Solvent Delivery System andWaters Nova-Pak C18 column, 300 mm×3.9 mm) at 37° C. Individualcompounds were eluted from the column with a gradient elution of 140 mMsodium acetate, 5.6 mM triethylamine, pH 5.2 (mobile phase A) and 60%acetonitrile (mobile phase B) with a flow rate of 1.0 ml/min. 9 Gradientconditions were as follows: initial=100% A, 2.0 min=90% A curve 11, 5.0min=86% A curve 11, 10.0 min=86% A curve 6, 18.0 min=73% A curve 6, 30.0min=60% A curve 10, 35.0 min=40% A curve 6, 39.0 min=10% A curve 6,followed by a wash with 100% B for 5 minutes and reequilibration for 5minutes at 100% A. BMAA peak identity was confirmed by comparison to acommercial standard (Sigma B-107; >94% pure) and was re-verified bymodified gradient elution. The concentration of BMAA in samples wasdetermined by the fluorescent tag using a Waters 2487 Dual-1Fluorescence Detector, with excitation at 250 nm and emission at 395 nm.Detection of the ACQ-derivatized BMAA was dependent on concentration andquantification was accomplished with comparison of equal amounts of BMAAand a norleucine internal standard (representing a single mid-rangeconcentration) resulting in a mean response of 51.2%. These dataexpressed the average response of values for several experiments anddepict the efficiency of the derivatization protocol and the relativeratio between BMAA and the internal standard. The results may have beenindicative of internal quenching of the derivativized compound, but didnot significantly affect sample quantification as the percentageresponse was consistent across the quantifiable concentration range. Thelimits of detection (LOD, defined as the lowest concentration of ananalyte in a sample that can be detected though not necessarilyquantitated) and limits of quantification (LOQ, the concentration withinthe linear range of the calibration curve relating absorbance toconcentration) were determined by a concentration gradient of anauthenticated standard (Sigma Chemical Co., St. Louis, Mo.). The LOD was0.00013 μmoles per injection and LOQ was 0.013 μmoles per injection forall analyses. For data interpretations, all sample analyses werequantified within the range of the LOQ or were reported as not detected(ND) (Table 1). Breeze scientific software (Trinity Consultants Inc.,Dallas Tex.) was used to control system operation and collect andanalyze data.

To confirm the identity of BMAA in HPLC fractions, TLC was carried outusing HPLC fractions and BMAA standards (BMAA, Sigma B-107; Methionine,Aldrich 15, 169-6). Briefly, 0.5 min HPLC fractions of derivatizedstandards and tissue samples were collected and pooled, and then wereconcentrated in a Savant speed-vac concentrator and spotted on channelsof TLC plates. TLC was carried out using glass-backed 250 μm analyticallayer silica gel plates (20×20 cm) with a mobile phase of 60 mlbutanol:15 ml glacial acetic acid:25 ml 0.5 N NaCl. After drying, theBMAA on plates were visualized with a 365 nm ultra-violet light.Finally, GC-MS of HPLC fractions containing peaks identified as BMAAconfirmed the presence of BMAA at 02.1 m/z for both the Sigma Standardcompound and a sample isolated from flying fox (bat) tissue (MVZ114607).

TABLE 2 BMAA in samples of cycads, cycad flour, and flying foxesConcentration Species Sample (μg/g) Cycas Gametophyte 240 micronesicaSarcotesta 9 outer integument 2,657 of sarcotesta Concentration (figig)*Based on Based on Kisby et al. Duncan et al. 1992 1990 Cycad seed Merizovillage 3 18 73 flour Agat village 8 1 4 Yigo village ND 5 8 Equivalentmean dose in kg of cycad flour Based on Based on Kisby et al. Duncan etal. Current 1992 1990 calculation Pteropus 114607 7,502 690 104 1,014mariannus 114606 1,879 173 26 254 (dried skin) 114609 1,287 118 18 174*Mean concentration reported, based on published values with reportedsample sizes ranging rom 1 to 4, found in Kisby et al., 1992, Neurology,42:1336:1340 and Duncan et al., 1990, Neurology 40: 767-772.

As shown in Table 2, flying fox skin tissue contained elevatedquantities of BMAA (1,287 to 7,502 μg/g), in contrast to the sarcotestaof cycad seeds which had a mean BMAA concentration of 9 μg/g. However,the outermost integument of the seed had extraordinarily highconcentrations of BMAA up to 2,657 μg/g. These results showing theabundance of BMAA in the fifty-year old museum specimens of flying fox(bat) tissues, demonstrated that the Chamorro people who consumed thisonce-abundant flying fox species unwittingly ingested high doses ofBMAA. For example, consumption of MVZ flying fox specimen #114607,assuming a fresh weight of 500 g and uniform distribution of BMAAthroughout the specimen, resulted in the ingestion of 3,751 mg of BMAA,which is comparable to consuming 1,014 kg of processed cycad flour.Table 2 further shows comparative values of BMAA concentrations in cycadflour samples from published reports. Differences in the values in Table2 reflect different extraction methods, differences in the analyticmethodology, and values that were not adjusted for efficiency of BMAArecovery.

Example 3 Cyanobacterial Neurotoxins: Cyanobacterial Origin of BMAA

BMAA was quantified in 200 mg samples of actively growing cyanobacteriaisolated from infected coralloid roots of Cycas micronesia Hill, cycadtissues; Azolla plants (collected near Hanapepe, Kauai); Gunnera plants(collected from Mt. Wailaleale, Kauai). All samples were homogenizedtwice in 0.1 N trichloroacetic acid and centrifuged at 15,800×g for 3min to precipitate proteins and extract free amino acids. Protein-boundBMAA was released by hydrolysis of the precipitate in 6N HCl undernitrogen for 24 hours, followed by centrifugation and ultrafiltration toremove sediment. An aliquot of the hydrolysis extract was thenfreeze-dried to complete dryness and resuspended in 20 mM HCl forderivatization. Sample extracts were derivatized with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and amino acidsquantified via HPLC separation as described above.

The presence of BMAA in the samples, as well as the identity and purityof the BMAA peak in the HPLC fractions, was verified by liquidchromatography mass spectroscopy (LC-MS) using an Agilent 1100 HPLCcoupled with a variable wavelength diode array detector (DAD) and an SLsingle quadrapole MS with an atmospheric pressure ionization source(API) using the electrospray ionization interface (ESI). Compounds wereseparated on a Waters SymetryShield RP 18 column heated at 30° C. with alinear gradient elution of CH3CN (10 40%) in water. Nitrogen gas waspurified and supplied to the ESI interface with a nebulizing pressure of35 psi and two distinct modes were used for detection of compoundswithin the MS. The DAD detected compounds at 254 nm with a full spectralscan from 200 600 nm and 0.5 nm resolution within a semi micro flowcell. The initial signal was determined in positive scan mode with a 100600 Da range at 50V fragmentor voltage, at a gain of 1.0 V. BMAA wasidentified through selective ion monitoring (SIM) in the positive ionmode with a dwell time of 45 msec and a 70 V fragmentor voltage. Forboth signals, the capillary voltage was 4 kV and the electron multipliervoltage gain was 4 V. The cycle time was 0.82 sec/cycle, split 50% foreach of the two MS signals.

Cyanobacterial Origin of BMAA

Cyanobacterial symbionts were isolated from infected coralloid roots ofCycas micronesica Hill, harvested from three accessioned specimens(vouchered specimens of known provenance) growing in the NationalTropical Botanical Garden, Kalaheo, Kauai, and grown as an axenicculture with repeated passages. For analysis of cycad root tissuesinfected with cyanobacterial symbiots, soil-borne bacteria were removedfrom root tissues of Cycas micronesica Hill by surface-sterilizing rootsimmersion in a solution of 70% ethanol for 3 min, followed by a 30 minimmersion in 1.6% sodium hypochlorite with 2 drops of surfactant and 3sequential washes with sterile deionized water. Surface-sterilized rootexplants (1-2 cm long) were excised and and cultured onto standard BG-11medium, pH 7.1 solidified with gellam gum (Sigma). Root explant cultureswere incubated in a controlled environment room with a 16 h photoperiodat a light intensity of 35-45 μmole/m²/s and temperatures of 25-30° C.After 7-10 days of culture, proliferation of colonies of thecyanobacterial symbiont of the root explants was clearly visible. Serialsubculture of individual cyanobacterial colonies ensured the absence ofresidual BMAA from root tissue. To assess the effects of amino acids oncyanobacterial growth, BG-11 medium was supplemented with glutamate orglutamine (0, 126, or 250 μmol/L); cyanobacterial growth was increasedtwo-fold by supplementation with these amino acids. Histologicalverification of culture purity was conducted prior to chemical analyses.The cyanobacterial colonies appeared to be generally devoid ofheterocysts, and prolific filamentous growth was observed.

Results. BMAA was not detected in non-infected cycad roots, but wasabundant in coralloid roots infected by the cyanobacterial symbiontNostoc, where coralloid roots with mild infections had 37 μg/g BMAA andcoralloid roots with heavy infections had 2 μg/g BMAA. Axenic culturesof Nostoc isolated from coralloid roots were found to have 0.3 μg/g ofBMAA. In non-root cycad tissues, BMAA is concentrated in cycad seeds(which are eaten by flying foxes), with 9 μg/g BMAA in the fleshysarcotesta, and up to 2,657 μg/g BMAA in the outer integument layer ofthe sarcotesta. See also, results in Table 1.

Additional studies of two unrelated plant species with cyanobacterialsymbionts were carried out to determine that cyanobacterial symbiontswere a source of BMAA in plant hosts. Azolla filiculoides, a floatingfern in rice paddies with a cyanobacterial symbiont, had 2 μg/g of BMAA.Gunnera kauaiensis, a large-leafed angiosperm with a cyanobacterialsymbiont, had 4 μg/g of BMAA in petiolar tissue. Levels of BMAA in theprotein pellet (protein-bound BMAA) were about 240-fold higher than thelevel fo BMAA quantified as free amino acid. These results confirmedthat BMAA of cyanobacterial origin may be found in many environments andmay be ingested in many food chains.

Example 4 BMAA in Brain Tissues

BMAA levels were measured in 200 mg samples of the superior frontalgyrus of brains of eight (8) Chamorro patients in Guam, and fifteen (15)Canadian patients. Tissues were provided by Dr. Patrick McGeer of theUniversity of British Columbia, Vancouver, B.C., Canada. Autopsiedtissues from patients were fixed in paraformaldehyde prior to storage in15% buffered sucrose maintenance solution, where the time intervalbetween death and autopsy varied from 4 hours to 5 days. Familialrelationships, clinical histories, and histochemical characteristics ofthe Chamorro patients had been disclosed previously (McGeer et al, 1997,Neurology 49, 400-409). In addition, autopsied tissued from Canadianpatients was provided, including two samples from two (2) Canadianpatients who were clinically diagnosed with Alzheimer's disease prior todeath, and thirteen (13) Canadian patientswho died of natural causesother than progressive neurodegenerative diseases.

Tissues were homogenized twice in 0.1 N trichloroacetic acid andcentrifuged at 15,800×g for 3 min to precipitate proteins and extractfree amino acids. Protein-bound BMAA was released by hydrolysis of theprecipitate at 110° C. in constant boiling 6N HCl for 24 hours.Particulate matter was removed from a 500 μl aliquot by ultrafiltration(Ultrafree-MC, Millipore Corp.) at 15,800×g and the resulting extractwas freeze-dried. Amino acids were resuspended in 20 mM HCl, applied toa Sep-Pac C₁₈ cartridge equilibrated with sequential washes of 100%methanol, 50% methanol in water, and a gradient of boratebuffer:acetronitrile (0.5M borate:0-60% CH₃CN) at 20% increments. BMAAin sample extracts was derivatized with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate following standardizedprotocols (Banack et al., 2003, Neurology 61: 387-389). Amino acids werequantified via HPLC separation as described above (Example 3). Thepresence of BMAA in the samples, as well as the identity and purity ofthe BMAA peak in the HPLC fractions, was verified by liquidchromatography mass spectroscopy (LC-MS) using an Agilent 1100 HPLCcoupled with a variable wavelength diode array detector (DAD) and an SLsingle quadrapole MS with an atmospheric pressure ionization source(API) using the electrospray ionization interface (ESI). Compounds wereseparated on a Waters SymetryShield RP 18 column heated at 30° C. with alinear gradient elution of CH3CN (10 40%) in water. Nitrogen gas waspurified and supplied to the ESI interface with a nebulizing pressure of35 psi and two distinct modes were used for detection of compoundswithin the MS. The DAD detected compounds at 254 nm with a full spectralscan from 200 600 nm and 0.5 nm resolution within a semi micro flowcell. The initial signal was determined in positive scan mode with a 100600 Da range at 50V fragmentor voltage. BMAA was identified using theextracted ion chromatogram in which the molecular ion peak wasconfirmed.

Results. As shown in Table 3, high levels of protein-bound BMAA(149-1190 μg/g) were found in frontal cortex tissue of all six Chamorrowpatients who had died from ALS-PDC. Frontal cortex tissue from five ofsix Chamorro patients who had died from ALS-PDC also had high levels offree BMAA (3-10 μg/g). In addition, significant amounts of free andprotein-bound BMAA was found in one asymptomatic Chamorro patient whodid not die of ALS-PDC, consistent with previous findings of Chamorroswho exhibited no clinical manifestations of ALS-PCD, but who showedsignficant neuroanatomical pathologies when autopsied. Significantconcentrations of BMAA were found in the frontal gyrus of brain cortexof two Canadian patients who were diagnosed as having died fromAlzheimer's disease. Frontal cortext tissues of the other thirteenCanadian patients, all of whom died of other causes, did not havedetectable levels of BMAA.

TABLE 3 Quantification of free and protein-associated BMAA in superiorfrontal gyrus tissue of Chamorro and Canadian patients FreeProtein-bound Age at BMAA BMAA Clinical Diagnosis at Death NationalityDeath Gender (μg/g) (μg/g) PDC (Lytico-bodig) Chamorro 60 M ND 1190 PDC(Lytico-bodig) Chamorro 69 M 6.7 644 ALS (Lytico-bodig) Chamorro 68 F10.1 610 PDC (Lytico-bodig) Chamorro 77 M 7.0 736 PDC (Lytico-bodig)Chamorro 60 M 9.1 149 PDC (Lytico-bodig) Chamorro 67 F 3.3 433Asymptomatic Chamorro 41 M 4.8 82 Asymptomatic Chamorro 61 M ND NDAlzheimer's Canadian — — 3.4 220 Alzheimer's Canadian — — 9.7 264Metastic cancer Canadian 39 F ND ND Heart failure Canadian 62 M ND NDCancer of the esophagus Canadian 69 M ND ND Chronic obstructive Canadian80 M ND ND pulmonary disease Lymphoma Canadian 60 F ND ND Cancer of thethyroid Canadian 86 F ND ND Heart failure Canadian 89 M ND ND Cancer ofthe pancreas Canadian 76 M ND ND Chronic obstructive Canadian 89 M ND NDpulmonary disease Heart failure Canadian 71 F ND ND Acute heart attackCanadian 80 F ND ND Chronic heart failure Canadian 87 F ND ND Aorticaneurysm Canadian 85 F ND ND

Various modifications can be made to the preferred embodiments withoutdeparting from the spirit and scope of the invention as defined in theappended claims.

1. A method of screening a subject having or at risk of having amyotrophic lateral sclerosis (ALS) comprising, obtaining a tissue sample from the subject and analyzing the sample to determine the amount of protein-bound β-N-methylamino-L-alanine (BMAA), wherein the presence of BMAA in the sample indicates the subject has ALS.
 2. The method of claim 1, wherein the protein-bound BMAA comprises BMAA incorporated into at least one protein.
 3. The method of claim 1, wherein the protein-bound BMAA comprises BMAA associated with at least one protein.
 4. The method of claim 1, further comprising determining the presence of free BMAA.
 5. The method of claim 1, wherein the subject has symptoms of ALS.
 6. The method of claim 1, wherein the subject is asymptomatic for ALS.
 7. The method of claim 1, wherein the subject has been identified as being at risk for developing ALS.
 8. The method of claim 1, wherein the tissue sample is neurological tissue.
 9. The method of claim 8, wherein the neurological tissue is associated with the central nervous system (CNS).
 10. The method of claim 9, wherein the tissue is brain tissue.
 11. The method of claim 8, wherein the tissue is cerebral-spinal fluid (CSF).
 12. The method of claim 8, wherein the neurological tissue is associated with the peripheral nervous system (PNS).
 13. The method of claim 1, wherein the tissue is non-neurological tissue.
 14. The method of claim 13, wherein the tissue is keratinous tissue.
 15. The method of claim 14, wherein the tissue is hair.
 16. The method of claim 14, wherein the tissue is skin.
 17. The method of claim 14, wherein the tissue is nail.
 18. The method of claim 17, wherein the nail is a fingernail.
 19. The method of claim 17, wherein the nail is a toenail.
 20. The method of claim 13, wherein the tissue is non-keratinous tissue. 